Chromatography columns

ABSTRACT

A method for packing a chromatography column having an inlet port at one end and an outlet port at the other, comprises feeding a slurry or smoke of particulate bed material in a carrier fluid and applying a predetermined pressure to the bed material while allowing the carrier fluid to escape, wherein both the inlet and outlet ports are protected by porous bed-retaining means positioned apart to provide a space for the bed between them, at least a final portion of the slurry or smoke being fed through a filler tube into the space for the bed until the space is full and the bed extends back into the filler tube, predetermined pressure to the bed being applied via the slurry or smoke in the filler tube, and the filler tube then sealed being against relaxation of the bed pressure.

The invention relates to chromatography columns, and in particular to amethod for packing columns and also to columns specifically adapted forpacking by that method.

Chromatography columns typically comprise a tubular container packedwith a bed of fine particulate material, such as silica particlesusually of a size within the range 5-45 μm. Inlet and outlet ports areprovided respectively at either end to enable liquids to be passedthrough the packed column during use, and the particles of the bed areheld in place and prevented from disappearing down the ports by porousbed-retaining means, such as sintered frits or wire gauzes inserted overthe ports at the ends of the column.

A high resolving power in a column requires not only bed material havinga small particle size, but also very uniform and close packing of thatmaterial. In order to obtain those desirable characteristics, variousmethods have previously been proposed, which require the application ofpressure to a bed of material, usually by filling through a slurryreservoir tube of the same or larger diameter and applying an axialcompressive force to the material in the combined column and slurryreservoir before replacing the reservoir tube with bed-retaining meansand end fittings to complete the packed column. Very high pressuresappear to be desirable with such techniques, pressures greater than 700bar being currently favoured. Unfortunately for large diameterpreparative columns such very high pressures would requireuneconomically substantial engineering, and even in the narroweranalytical columns, while some of these known techniques can indeedproduce very good results in expert hands, results do still tend to bevariable.

We have now found that particulate bed materials can become resilientlycompressed during packing procedures. Thus for example, a bed uniformlycompressed under about 400 bar may then relax by 5-10% when the pressureis released, and in this way a compressed bed can relax at one end whenthe slurry reservoir is removed, leading to uneven packing.

According to one aspect of the present invention, a method for packing achromatography column having an inlet port at one end and an outlet portat the other, by feeding a slurry or smoke of particulate bed materialin a carrier fluid and applying a predetermined pressure to the bedmaterial while allowing the carrier fluid to escape, is characterised inthat the method comprises protecting both the inlet and the outlet portswith porous bed-retaining means positioned apart to provide a space forthe bed between them; thereafter feeding at least a final portion of theslurry or smoke through a filler tube into the space for the bed untilthe space is full and the bed exends back into the filler tube; applyingthe predetermined pressure to the bed via the slurry or smoke in thefiller tube, and sealing the filler tube against relaxation of the bedpressure. While the slurry or smoke is being fed through the fillertube, the carrier fluid is forced to drain away through one or bothporous bed-retaining means.

In general we prefer to use a slurry (i.e. using a carrier liquid)rather than a smoke (i.e using a gaseous carrier) because of the highflow rates needed to obtain adequate packing pressures when usinggaseous carriers.

According to a further aspect of the invention, a chromatography columnfor packing with particulate bed-material, comprises a tubular containerhaving at one end an end portion with an inlet port and at the other endan end portion with an outlet port, both ports being protected by porousbed-retaining means positioned with a space for the bed between them,characterised in that the column also has a filler tube whichcommunicates directly with the space for the bed.

In a preferred method and corresponding apparatus, the filler tube has adiameter sufficiently small to hold particulate material packed thereinagainst the predetermined pressure, whereby, on packing the space withthe bed material at the predetermined pressure and on packing also atleast an end portion of the filler tube adjacent to the bed with furtherbed material, the filler tube is sealed automatically against relaxationof bed pressure therethrough when the pressure applied via the slurry isreleased.

The narrower the bore of the filler tube, the more effective is thesealing against greater pressures. However its narrowness is limited bythe need to feed the slurry or smoke through it at a rate sufficient toprovide the predetermined pressure in the column. Its sealing efficiencycan also be affected by the physical nature of the particulate material,especially functional properties, the length of filler tube penetrationby the particulate material, and the ratio of filler tube diameter tocolumn diameter in the case of end cap filler tubes.

However, as a guide, for silica particles of 5-10 μm, a moderate sealagainst relaxation of 400 m bar pressure can be obtained with fillertubes of internal diameter about 6.4 mm, although narrower tubes arepreferred for more effective and secure sealing. Narrow filler tubes arealso preferred, especially when using narrow columns, so as to avoidsignificant loss of resolution due to sample migration into the fillertube. Filler tubes having cross-sectional areas less than 5% of thecolumn's cross sectional area, are preferred. However, the use ofinternal diameters less than about 0.25 mm tends to increase the risk ofblocking during filling.

As will be appreciated, a column using a narrow filler tube to obtainautomatic sealing against pressure relaxation at the end of the packingcycle, will also require sealing against loss of eluent and sampleliquids through the filler tube, before it can be used forchromatography purposes. This may be achieved by using a simple blankingmember on the exposed end of the filler tube. It is preferred,especially for filler tubes having internal diameters at the wider endof the effective range, to pack the whole of the filler tube, or atleast such portion as is to be retained during use of the column. Thisis to minimise the chances of a packed plug of the solid working free ofits compressed state when lubricated by eluent or sample liquids. Analternative is to insert a rod down the filler tube until it reaches thepacked plug at the column end, thereby to fill any space beyond the plugand prevent eluent or sample liquid permeating into it.

The position where the filler tube enters the column does not appear tomake a very significant difference to the efficiency of the column, andthey can therefore be positioned according to other criteria. For widebore columns, e.g. with internal diameters greater than about 25 mm, weprefer to introduce the filler tube by passing it through an end portionand through its bed-retaining means, preferably at the outlet end, so asto avoid weakening the column wall and reduce its ability to withstandhigh packing pressure. With narrower columns, however, where strength isless of a problem than finding room in the end cap for a filler tube inaddition to the inlet port, we prefer to introduce the filler tubethrough the wall of the tubular column.

We have referred above to a desireability for using relatively lowpacking pressures so as to retain a resilient compression of theparticles rather than to crush them into a state of collapse. Lowpacking pressure also enables thinner walled columns to be used, andthis can be considerably advantageous, especially with large diameterpreparative columns.

With the present method of packing columns for use at typical operatingpressures in the region of 35 bar (500 psi), we find that we canconsistently obtain high resolution columns (e.g. as typified by theExamples described hereinafter) when using only 400 bar pressures forpacking 5-10 μm silica. Indeed, our preferred packing pressures, atleast for column diameters 0.5 cm to 5 cm, lie within the range 300-500bar (4350-7250 psi). However, there is nothing in the method itselfwhich precludes the use of much higher pressures, where for example thenature of the particles make more extreme pressures desirable.

The column can be packed by a method in which all the slurry or smoke isfed through the filler tube into the space for the bed after both inletand outlet ports have been protected by the porous bed-retaining means,thereby progressively to fill up the space with particulate bed materialuntil it is full, while applying pressure via the slurry or smoke beingfed through the filler tube. In general we prefer that substantially allthe slurry which is fed through the feeding tube, be fed at thepredetermined packing pressure. As may be realised, when slurry at forexample, 400 bar is fed to an empty column, there will be an immediatedrop in pressure, but as soon as a layer of particulate material formsacross the bed-retaining means through which the liquid carrier escapes,this layer very quickly becomes capable of estabilishing a 400 barpressure drop across it, provided a suitably high output pump isemployed, and/or a high viscosity liquid is used for the slurry.

However, we find that an initial surge which occurs when feeding slurryinto an empty column, does not always produce the optimum packing. Weprefer to operate our process by placing an initial portion of theslurry directly into the column while a porous bed-retaining means isprovided at one end only, then completing assembly of the column andfeeding the remainder of the slurry through the feeding tube, preferablyat the predetermined packing pressure as described above. Generally weprefer to fill the column completely with slurry before completingassembly. Although some of the carrier liquid may drain through thebed-retaining means the bulk of it tends to remain until hit by theslurry from the feed pipe, and thereby forced through. One effect of theinitially added slurry appears to be a dissipation of the impact ofslurry suddenly forced out of the feeding tube into an empty columnunder pressure from a reservoir at 400 bar, with flow profiles in thebed avoided as pressure is maintained by slower, more consistent flowrates.

Columns which we have packed in this manner have been capable of givinghigh performance results (e.g. better than 50,000 plates/meter forcolumns even as short as 10 cm), but much of this potential performancecan be lost if other variable parameters of the column are not also of asimilar standard. In particular we find that the advantages of thepresent invention can be realised most markedly when the column isadapted for controlling the flow pattern of fluids flowing through theend portions of the bed. To this end we prefer a column having adistribution chamber between each port and its bed-retaining means,wherein to enable radial flow to occur with substantially lessresistance than radial flow within the bed. The chamber may suitably beformed by a woven wire gauze, positioned to space the porousbed-retaining means from its adjacent end of the column.

A further feature which we find may improve the resolution of a columnpacked using a filler tube according to the method of the presentinvention, is the provision at the outlet end of the column of aradially central impermeable blanking means between the bed retainingmeans and the outlet port, and spacing means to enable liquids flowingaround the periphery of the blanking means during use to reach theoutlet port. We find that a spacing means comprising a woven wire gauzecan provide good support for the bed retaining means and blanking meansagainst the bed packing pressure, while offering only a low resistanceto liquids flowing radially inwards to the port. Possible alternativesinclude sinter discs and endcaps relieved to provide an array of pointsupports, but in general we prefer to use the woven wire gauzes.

The blanking means can be a thin circular disc located between the bedretaining means and spacing means, with a surrounding porous annulus tohold the disc central. A preferred form is a woven gauze with theradially central area blanked off by a disc of impermeable material suchas PTFE embedded in the gauze. With some forms of bed-retaining meansand spacing means, e.g. sinter discs, it is possible for the blankingmeans to be incorporated therein as an integral part, e.g. by embeddinga PTFE disc directly into the surface. However this form of constructionis only appropriate where it still leaves a porous bed retaining layeroffering low resistance to radial flow of liquid between the bed andblanking means, and a porous spacing layer offering low resistance toradial flow of liquid between the blanking means and outlet port. PTFEembedded in a woven gauze tends to block such radial flow, and so whenusing woven gauzes for both bed retaining means and spacing means, weprefer that the blanking means be a separate layer (e.g. a PTFEimpregnated third gauze) located between those two gauzes.

A similar structure incorporating a blanking means can also be used withadvantage at the inlet end, but the beneficial effect of such astructure is generally less noticable at the inlet end than at outletend. In either case we prefer that the radius of the impermeable area ofthe blanking means be about two thirds the radius of the porous bedretaining means.

The invention is illustrated by reference to three specific embodimentsthereof, shown in the accompanying drawings, in which:

FIG. 1 is a foreshortened part through a wide bore preparatory column,

FIG. 2 is a section through the inlet region of an alternativepreparatory column, and

FIG. 3 is a section through a narrower analytical column.

Each of the three drawings is a diagrammatic representation in that theyare not entirely to scale, some dimensions having been exaggeratedslightly for clarity and simplicity of illustration.

In FIG. 1, the column comprises a thickwalled stainless steel cylinder1, across the ends of which are bolted end caps 2, 3 to complete atubular container forming the basis of the column. Immediately withinthe end caps at both ends are two woven wire gauzes, those 4, 5 next tothe end caps being of a coarse mesh to act as liquid distributors, andthe inner gauzes 6, 7 being of a much finer mesh. These latter are thebed-retaining means, and as such must be sufficiently fine to preventthe bed particles from passing through while remaining porous to thevarious liquids used during packing and operation. (Where the mesh sizedifferential is excessive, a further gauze of intermediate mesh size maybe placed between them). An impermeable blanking disc 8 is also providedbetween the gauzes at the outlet end, to control the flow of liquidthrough the column.

Passing through one of the end caps 3, and terminating flush with itsinner surface is an outlet tube 9 mounted in a stud coupling 10, toprovide an outlet port 11, in conventional manner. Passing through theother end cap 2 is a further stud coupling 12, but this has beenmodified by combining it with a T coupling. This also carries an inlettube 13, providing an inlet port 14. Threaded through the coupling 12 isa feeding tube 15, which extends inwards below the level of the end cap,and passes through both gauzes 4, 6 to end in a boss 16. Between theboss and the gauzes is placed a polytetrafluoroethylene (PTFE) sealingwasher 17.

The feeding tube extends out of the T coupling to a tensioner whichtensions it outwards so as to provide a good seal at the PTFE washer.The tensioner consists of a split tube clamp 18 with a spring 19 undercompression and biasing apart the clamp 18 and the T coupling 12. Thefeeding tube is sealable by a cap 20 screwed onto the end of the clamp18.

The column is packed by assembling the thickwalled cylinder 1, lower endcap 3 with its outlet port 9, gauzes 5, 7, and blanking disc 8; thisassembly then being filled with a slurry of the bed material in acarrier liquid. The upper end cap 2, complete with its gauzes 4, 6 andinlet port assembly, is then clamped to the top of the thickwalledcylinder. With the cap 20 removed, the filler tube is connected to aslurry reservoir maintained at the predetermined pressure by a pump, andthe slurry is caused to flow through the filler tube into the column. Asthe carrier liquid of the slurry starts to be forced through thebed-retaining gauzes, a layer of a particulate bed material quicklyforms, slowing up the flow of liquid as a pressure gradient equal to thepredetermined pressure is developed across it. When the column becomesfull, the rate of liquid flow drops dramatically as the much narrowerbore filler tube starts to become packed. Packing is preferablycontinued at this slow rate until the filler tube becomes packed to thetop of the clamp 18. The pressure in the reservoir is then released andthe clamp disconnected from it. The particulate solid within the fillertube retains the packing pressure within the solid particles in thecolumn, but the cap 20 requires replacing so as to retain eluent andsample liquids passed through the column during chromatographic usage.The long filler tube 15 shown, made possible the convenient illustratedway of sealing it using the PTFE washer 17. However, simply forretaining the packing pressure, a very much shorter tube may be usedsuccessfully, especially where there is a large difference in diameterbetween the filler tube and the column.

We have used this type of structure with thick-walled cylinders of 50 mminternal diameter, and the inlet assembly comprising a 3.5 mm OD inlettube and 1.8 mm OD filler tube giving a clearance of a 0.13 mm betweenthem for passage of eluent and sample liquids during chromatographicuse. The internal diameter of the feeding tube was 0.75 mm. With acolumn length between bed-retaining gauzes of 100 mm and using a slurryof 150 g of 5 μm average diameter silica particles in 600 ml of a 50/50mixture of glycerol and methanol by volume, various packings at 400 barby the above method consistently gave columns having resolutions ofbetween 50,000 and 120,000 plates per meter, with near theoretical peakshapes.

In FIG. 2 only a portion of an alternative fitting is shown. A finewoven gauze 21 for retaining the bed extends across the end of thecolumn and is clamped around its perimeter, substantially as shown forthe corresponding gauze 6 of FIG. 1. Behind this is a second gauze 22 ofsimilar fine weave, but of smaller diameter, typically 1 to 1.5 cmacross. In its upper surface around the perimeter is embedded a PTFEannulus 23 forming a seal against the end cap 24 of the column, throughwhich is drilled an inlet port 25. A filler tube 26 passes upwardsthrough the inlet port, a PTFE washer 27 providing a seal where thefiller tube passes through the gauzes. At the end of the filler tube isa radial flange 28, and around the tube is forced a short length of asimilar tube 29, also having a radial flange 30. The short outer tube isa tight but slideable fit onto the filler tube, typically having aninternal diameter about 50 μm (0.002 inch) smaller than the filler tubeoutside diameter before assembly. The two gauzes are sandwiched firmlybetween the two flanges, and between the upper flange 30 and the end cap24 is a small spacer washer 31 also formed from the fine woven gauze.The filler tube assembly is initially held up to the end cap simply byconventional retaining nuts (not shown), avoiding the more complexbiasing device needed in FIG. 1.

The column of FIG. 2 is packed by feeding a slurry containing bedparticles through the filler tube 26 (substantially as described forFIG. 1), but in this design the hole through the gauzes for the fillertube is sealed against loss of bed particles to the inlet port by theclamping action of the two flanges 28, 30, the sealing washer 27, thetightness of the short tube 28 around the filler tube, and in particularby the packing pressure pressing the assembly against the end cap.

During use of the packed column, fluid (e.g. element or sample solution)is pumped into the column via the inlet port 25, passing through thespacer gauze 31, into the two main gauzes 21, 22. The full width gauze21 serves not only to retain the bed, but also to provide a relativelylow resistance to radial flow of the fluid, thereby distributing thefluid across the top of the bed. The other gauze 22 of smaller diameterfilters out any solids which might block the larger gauze, and the areaof contamination is limited by the PTFE/sealing annulus 23, therebymaking possible deblocking of the filler gauze by reverse flow. Stresseson the thin gauzes might be relieved to some extent by stepping themouth of the inlet port to receive the upper flange and spacing gauze,but in practice the arrangement as shown has not so far failed usthrough gauze failure due to their bending as illustrated.

The size of the dead area produced in the shadow of the filler tube cangenerally be reduced still further by providing near the end of thetube, a shoulder (to replace the short flange tube 29), and swaging overthe ends of the tube to grip the gauzes against the shoulder. The flange28 is then not required, and the gauzes can be mounted on the end of thetube from the column end, before swaging.

FIG. 3 shows an assembly more suited to narrower columns of, forexample, 13 mm ID. This consists essentially of a conventional column towhich has been added a side arm. The column comprises a stainless steelcylinder 41 with end caps 42, 43 retaining loosely woven spacer gauzes44, 45 (for allowing liquid distribution), and more tightly wovenbed-retaining gauzes 46, 47. Midway between the ends is a narrower (e.g.1.8 mm OD) filler tube 48 brazed into the cylinder 41 so as tocommunicate with the space for the bed inside it.

The packing procedure is essentially the same as that described abovefor the larger apparatus, and we have packed 13 mm columns in thismanner using 600 bar packing pressures on 5 μm average diameter silicaparticles, and obtained consistently resolving powers better than 50,000plates per meter.

One of the features we have found of this method of packing, is theconsistency of the results. Provided that the filler tube is ofsufficiently narrow bore to hold the packing pressure securely (e.g. a0.75 mm for conditions as described above), the operation becomesautomatic the operator has considerable experience or only limitedexperience at column packing.

I claim:
 1. A method for packing a chromatography column having an inletport at one end and an outlet port at the other end, said methodcomprising the steps of:(a) forming particulate material into a bedbetween porous bed-retaining means located to protect the inlet andoutlet ports by feeding a slurry or smoke of particulate bed material ina carrier fluid into the column; (b) applying a predetermined pressureto the bed of particulate material while allowing the carrier fluid toescape; (c) thereafter feeding at least a final portion of the slurry orsmoke of particulate material through a filler tube in communicationwith the space in the column until the space is full; and (d) continuingto feed the at least final portion of the slurry or smoke of particulatematerial until the particulate material bed extends back into the fillertube to thereby maintain the predetermined pressure applied to the bedvia the slurry or smoke of particulate material to thus seal the fillertube against relaxation of the pressure applied to the bed ofparticulate material.
 2. A method as claimed in claim 1 wherein step (a)is practiced by the steps of:(i) feeding an initial portion of theslurry or smoke of particulate material into the column while a porousbed-retaining means is provided at one end only, and (ii) assembling aporous bed-retaining means at the other end of the column, whereby theremaining final portion is fed through the filler tube.
 3. A method asclaimed in claim 1 wherein all the slurry or smoke is fed through thefiller tube into the space for the bed after both inlet and outlet portshave been protected by the porous bed-retaining means, therebyprogressively to fill up the space with particulate bed material untilit is full, while applying pressure via the slurry or smoke being fedthrough the filler tube.
 4. A method as claimed in any one of thepreceding claims wherein the particulate bed material is fed to thespace in a carrier liquid, in the form of a slurry.
 5. A method asclaimed in claim 1 wherein the filler tube has a diameter sufficientlysmall to hold particulate material packed therein against thepredetermined pressure, whereby, on packing the space with the bedmaterial at the predetermined pressure and on packing also at least anend portion of the filler tube adjacent to the bed with further bedmaterial, the filler tube is sealed automatically against relaxation ofbed pressure therethrough when the pressure applied via the slurry isreleased.
 6. A method as claimed in claim 1 or 5 wherein thepredetermined packing pressure lies within the range 300-500 bar.
 7. Achromatography column for packing with particulate bed-material, saidcolumn comprising a tubular container having a first end portiondefining an inlet port at one end and a second end portion defining anoutlet port at the other end, both said inlet and outlet ports beingprotected by porous bed-retaining means positioned with a space for thebed between them, and filler tube means which communicates directly withthe space for the bed for feeding at least a final portion of a slurryor smoke of particulate material from a pressurized source thereof untilsaid space is full of particulate material and said particulate materialextends at least partially into said filler tube means to maintain saidbed at a predetermined pressure when said filler tube means isdisconnected from the pressurized source to thereby prevent pressurerelaxation of said bed.
 8. A chromatography column as claimed in claim 7wherein the filler tube has an internal diameter equal to or less than6.4 mm.
 9. A chromatography column as claimed in claim 7 or claim 8,wherein the filler tube is introduced into the column through one end,passing through an end portion and then through one of the bed-retainingmeans to reach the space for the bed.
 10. A chromatography column asclaimed in claim 7 or claim 8, wherein the filler tube communicates withthe space for the bed by passing through the tubular container at aposition between the two bed retaining means.
 11. A chromatographycolumn as claimed in claim 7 and having a distribution chamber betweeneach port and its porous bed-retaining means, through which to enablefluid to flow within the bed.
 12. A chromatography column as claimed inclaim 11, wherein the chamber is formed by a woven wire gauge positionedto space the porous bed-retaining means from its adjacent end of thecolumn.
 13. A chromatography column as claimed in claim 7 wherein thebed-retaining means which protects the outlet port has a radiallycentral region which is non-porous.
 14. A chromatography column asclaimed in claim 13 wherein the bed-retaining means which protects theoutlet port comprises a woven wire gauge in which the radially centralregion is impregnated with a non-porous material.
 15. A chromatographycolumn comprising:container means having opposing ends for containing abed of particulate therein; means defining an inlet port at one end ofsaid container means; means defining an outlet port at the other end ofsaid container means; bed-retaining means positioned at each of saidopposing ends to define therebetween a space for the particulatematerial bed and for retaining the particulate material in said definedspace; filler tube means in fluid communication with said space andconnectable to a pressurized source of a slurry or smoke of particulatematerial for feeding at least a final portion of a slurry or smoke ofparticulate material from the pressurized source thereof into said spaceuntil said space is full of particulate material and said particulatematerial extends at least partially into said filler tube means to sealsaid bed of particulate material in said bed by virtue of saidparticulate material extending at least partially into said filler tubemeans and thus to maintain said bed at a predetermined pressure whensaid filler tube means is disconnected from said pressurized source tothereby prevent pressure relaxation of said bed.
 16. A chromatographycolumn as in claim 15 wherein said filler tube means is elongated andincludes:means to support said filler tube means coaxially with saidinlet port; seal means fixed to a distal end of said filler tube meansfor sealing said inlet port to prevent escape of particulate materialtherethrough; and biasing means for biasing said seal means into sealingengagement with said inlet port.
 17. A chromatography column as in claim16 wherein said filler tube means further includes liquid seal means ata proximal end of said filler tube means for retaining liquids in thecolumn during use.